4.1- Effect of BHRF-1 on cell physiology
4.1.1- Cultures with non-controlled pH and pO2conditions: Shake flasks cultures
After engineering KB26.5 hybridoma cell lines with BHRF-1, the transformed cell population was kept under selective pressure for several passages. Protective effect of BHRF1 against apoptosis was studied and described previous to the present work (Juanola et al., 2009). Then, differences in terms of cell physiology between the parental cell line KB26.5 and the engineered KB26.5-BHRF1 were observed after selecting the engineered cell population. The main physiological parameters were initially determined in shake flask cultures. Time profiles for cell growth, cell viability, glucose and lactate concentrations for both cell lines (A1 for KB26.5 and B1 for KB26.5-BHRF1) are displayed in Figure 1 . Napierian logarithm of viable cells versus time is plotted to better understand the changes in cell growth rate, as it will be discussed further down (A2 for KB26.5 and B2 for KB26.5-BHRF1). Considering that the experiments were carried out in parallel by duplicate (same time points), the mean values are plotted together with the standard deviation.
The parental KB26.5 cell line reached a cell density about 1.79±0.3·106 cells/mL after 72 hours of culture, when cell viability started to decrease sharply. Concerning glucose and lactate concentration, high consumption and production rates respectively could be deduced by the sharp decay of the concentration profiles. Parallel to “plateau” of the cell growth profile, glucose consumption and lactate production also indicates a metabolic arrest at 72 h of culture, since their concentration values become almost constant. Cell viability decays drastically right after the cell density peak (maximum cell density), reaching values about 50% at 96 hours (24 hours after the cell density peak).
The growth, viability, glucose and lactate profiles obtained from KB26.5-BHRF1 cultures were absolutely different than those for the parental cell line. Cell culture expansion was much faster, and maximum cell density reached was about 3.92±0.03·106 cell/mL, what represents a 2-fold increase in respect to the parental cell line. Also, very different was the cell viability profile after the cell density peak, since cell viability was kept over 90% for 24 hours after the maximum of cell density, indicating the delay on the apoptosis due to the effects of BHRF1. Glucose and lactate profiles showed an initial phase of glucose consumption and lactate generation; glucose concentration profile decrease was similar to the obtained in KB26.5 culture, but lactate production rates were clearly lower being reduced by almost half. However, since cell density was almost doubled, the specific glucose consumption rate was clearly affected by BHRF1 expression and, consequently, the lactate production. In order to better quantify the differences, Table 1 complies the main physiological parameters for both cell lines.
In general terms, the expression of BHRF1 was beneficious for cell physiology and growth. Cell growth rate was increased by about 78%, but also glucose consumption specific rate was decreased by 15% and lactate production reduced to more than 50%. Overall resulted in a more efficient usage of the main carbon source. This fact is translated in a more than 2-fold increase in total cell density reached using the same media and culture conditions.
4.1.2- Bioreactor cultures with controlled pH and pO2conditions
In order to confirm the beneficial effect of BHRF1 on cell physiology (on cell growth and nutrients usage), cultures of both cell lines were performed in 2-Litres bioreactor. Bioreactor cultures should not only discard that the differences encountered before were related to changing culturing conditions when culturing cells in shake flasks, but also to generate enough data to feed the metabolic model and perform the Flux Balance Analysis. In addition, product concentration (IgG3) was measured from the moment the cells reached the maximum cell density. Figure 2 shows the time profile of cell density and viability; glucose, lactate and product concentrations evolution. As the experiments were carried out in the same bioreactor by duplicate but not in parallel (different time points), both replicates are plotted for each cell line, showing comparable behaviors. Napierian logarithm of viable cells versus time is plotted for each replicate inFigure 3 , to better understand the changes in cell growth.
In general terms, the results obtained in 2-Litres bioreactors are consistent with those previously observed in shake flask cultures. Maximum of cell density was slightly improved for KB26.5, reaching values about 2.34 ± 0.39·106 cell/mL, probably due to the pH control. Even tough lactate was produced at similar rates, the lack of pH control in the shake flaks probably negatively affected cell growth, situation reverted in the bioreactor cultures. In the case of KB26.5-BHRF1, the maximum cell density observed was significantly increased, obtaining 3.93 ± 0.01·106 cell/mL. In addition, the viability strongly decayed after 48h for KB26.5, and it was maintained above 90% the entire culture for BHRF1-KB26.5.
Despite the concordance with the results obtained in shake flask, a difference was observed since glucose was completely depleted in KB26.5 culture, indicating the extension of the culture time due to the pH control, what could have an effect on cell density increase. To further compare the performance of both strains in Bioreactor, main physiological parameters were calculated and complied in Table 2 .
Similarly to shake flask culture results, cell growth was improved by more than 30% in KB26.5-BHRF1, and also glucose consumption and lactate generation rates were reduced 42% and 54% respectively. Therefore, differences between strains should be related to the effects of BHRF1 on cell physiology instead of being a consequence of the culturing conditions. Due to the extension of the growth phase, KB26.5-BHRF1 increased the final product concentration by 3.7 times. The specific and volumetric productivity were also increased by 2 and 3 times respectively.
Finally, and before preforming Flux Balance Analysis (FBA), the specific consumption rates for all the metabolites measured in bioreactor experiments were calculated in the exponential growth phase (Table 3 ). Values have been calculated as the mean of the data obtained by duplicates.
Again, the results coincide in terms of lower specific consumptions rates for glucose and for almost all amino acids, as well as lower production rates of by-products, what might be a signal of a more efficient cell metabolism, somehow mediated by the expression of BHRF1. It should be emphasized the significant reduction in glutamine consumption and, therefore, in the ammonia generation, which is well known to be a toxic by-product in mammalian cell cultures (Yang and Butler, 2000), obtaining a double benefit of lactate and ammonia reduction in culture. Alanine generation was also significantly reduced in KB26.5-BHRF, taking into account that is an amino acid that it is often accumulated in mammalian cell cultures (Quek et al., 2010). In any case, a metabolic flux balance is required to evaluate and discuss the possible effects of the protein BHRF1 on the general behavior of the engineered line in respect to the parental cell line. Interestingly, the expression of BHRF1 has an effect on such metabolism improving the efficiency of nutrients usage needed for cell growth (see Table 1, 2 and 3 ), the reduction of by-products, as well as, the increase of cell growth.